The long term goal of our research is to understand the mechanisms of inward-rectifier K+ (Kir) channels, a group of highly specialized integral membrane proteins. These channels can pass K+ ions in the inward direction more efficiently than in the outward direction, a property referred to as inward rectification. Such a property allows the channels to maintain and regulate cell resting membrane potential and thereby accomplish many important physiological tasks. The experiments proposed here are specifically aimed at understanding the mechanisms underlying the rectification, i.e., how the channels are blocked by intracellular cationic blockers with an exceedingly strong voltage dependence: a valence of up to five. Based on our extensive mechanistic and quantitative studies of the Kir2.1 channel, we proposed that the exceedingly sharp rectification primarily reflects the movement of up to five K+ ions, displaced by an intracellular blocker, across the electric field that drops steeply across the narrow K+ selectivity filter. Our proposal in turn poses the following three important and experimentally testable hypotheses: 1) strong depolarization will energetically uncouple the blocker from K+ ions in the pore and thereby eliminate the associated voltage dependence; 2) the cytoplasmic pore of Kir harbors monovalent permeant cations; and 3) a narrow gasket-like constriction at the inner end of the pore prevents K+ ions from bypassing the blocker, thus ensuring outward displacement of K+ ions by the intracellular blocker and thereby producing strong voltage dependence. To test the above hypotheses, and an additional one regarding how mutations of certain critical channel residues alter some fundamental pore properties, we will express Kir channels in Xenopus oocytes and quantitatively examine their functional properties in mutagenesis-based electrophysiological studies, which will be further aided with more direct structural techniques. The proposed studies have important medical implications because Kir channels play many important physiological roles, such as controlling the heart rate, modulating neural transmission, and coupling blood glucose levels to insulin secretion. They represent therefore important pharmacological targets for medical intervention in various disease states.